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Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

Holland SJ, Liao XC, Mendenhall MK, Zhou X, Pardo J, Chu P, Spencer C, Fu A, Sheng N, Yu P, Pali E, Nagin A, Shen M, Yu S, Chan E, Wu X, Li C, Woisetschlager M, Aversa G, Kolbinger F, Bennett MK, Molineaux S, Luo Y, Payan DG, Mancebo HS, Wu J - J. Exp. Med. (2001)

Bottom Line: Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade.In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl.Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

View Article: PubMed Central - PubMed

Affiliation: Rigel, Incorporated, South San Francisco, CA 94080, USA. sholland@rigel.com

ABSTRACT
In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

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The NH2-terminal myristoylation site and COOH-terminal unique regions of SLAP-2 are required for inhibition of antigen receptor signaling. Epitope-tagged wild-type SLAP-2, SLAP-2-myr, and SLAP-2-ΔC in the pTRA-IRES.GFP vector were stably introduced into tTA-BJAB or tTA-Jurkat cells, and GFP-positive cells were enriched by sorting. Induced surface CD69 expression was analyzed in vector-infected cells (dotted line) and cells infected with wild-type or mutant SLAP-2 (solid line) with analytical-gating on GFP expression. The geometric means of APC-CD69 fluorescence is shown. (A) tTA-BJAB cells stimulated with anti-IgM F(ab′)2.Wild-type (WT) SLAP-2 reduced anti-IgM F(ab′)2-induced CD69 expression by ∼2-fold, whereas SLAP-2-myr and SLAP-2-ΔC resembled the vector control. Percentage of GFP positive cells: vector 91%; wild-type SLAP-2 89%; SLAP-2-myr 80%; SLAP-2-ΔC 87%. (B) tTA-Jurkat cells stimulated with C305. Wild-type SLAP-2 reduced C305-induced CD69 expression by ∼3-fold, whereas inhibition was compromised in cells expressing SLAP-2-myr and SLAP-2-ΔC. Percentage of GFP-positive cells: vector 84%; wild-type SLAP-2 46%; SLAP-2-myr 44%; SLAP-2-ΔC 45%. (C) Equal aliquots of infected tTA-BJAB cells were lysed and analyzed by anti-FLAG Western blot for SLAP-2 protein expression. (D) Equal aliquots of infected tTA-Jurkat cells were lysed and analyzed by anti-FLAG Western blot for SLAP-2 protein expression. (E) Membrane pellet (P) and soluble fraction (S) of wild-type SLAP-2 and SLAP-2-myr–infected tTA-BJAB cells were immunoprecipitated (top panel) or loaded directly (center bottom panels) and immunoblotted with antibodies raised against the FLAG epitope (top panel), the integral membrane protein CD40 (center panel), and the cytoplasmic protein JNK (bottom panel). A fraction of wild-type SLAP-2 but not SLAP-2-myr was localized in the membrane fraction.
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fig5: The NH2-terminal myristoylation site and COOH-terminal unique regions of SLAP-2 are required for inhibition of antigen receptor signaling. Epitope-tagged wild-type SLAP-2, SLAP-2-myr, and SLAP-2-ΔC in the pTRA-IRES.GFP vector were stably introduced into tTA-BJAB or tTA-Jurkat cells, and GFP-positive cells were enriched by sorting. Induced surface CD69 expression was analyzed in vector-infected cells (dotted line) and cells infected with wild-type or mutant SLAP-2 (solid line) with analytical-gating on GFP expression. The geometric means of APC-CD69 fluorescence is shown. (A) tTA-BJAB cells stimulated with anti-IgM F(ab′)2.Wild-type (WT) SLAP-2 reduced anti-IgM F(ab′)2-induced CD69 expression by ∼2-fold, whereas SLAP-2-myr and SLAP-2-ΔC resembled the vector control. Percentage of GFP positive cells: vector 91%; wild-type SLAP-2 89%; SLAP-2-myr 80%; SLAP-2-ΔC 87%. (B) tTA-Jurkat cells stimulated with C305. Wild-type SLAP-2 reduced C305-induced CD69 expression by ∼3-fold, whereas inhibition was compromised in cells expressing SLAP-2-myr and SLAP-2-ΔC. Percentage of GFP-positive cells: vector 84%; wild-type SLAP-2 46%; SLAP-2-myr 44%; SLAP-2-ΔC 45%. (C) Equal aliquots of infected tTA-BJAB cells were lysed and analyzed by anti-FLAG Western blot for SLAP-2 protein expression. (D) Equal aliquots of infected tTA-Jurkat cells were lysed and analyzed by anti-FLAG Western blot for SLAP-2 protein expression. (E) Membrane pellet (P) and soluble fraction (S) of wild-type SLAP-2 and SLAP-2-myr–infected tTA-BJAB cells were immunoprecipitated (top panel) or loaded directly (center bottom panels) and immunoblotted with antibodies raised against the FLAG epitope (top panel), the integral membrane protein CD40 (center panel), and the cytoplasmic protein JNK (bottom panel). A fraction of wild-type SLAP-2 but not SLAP-2-myr was localized in the membrane fraction.

Mentions: Myristoylation has been shown to localize signaling proteins including Src family kinases to the membrane compartment and in many cases to be critical for their function (29). As SLAP-2 contains a putative NH2-terminal consensus motif (MGX1–4T/S) for addition of a myristoyl group, we generated an amino acid substitution in the myristoylation motif within a FLAG-tagged full length SLAP-2 (SLAP-2-myr) and examined its effect on antigen receptor–induced signaling. SLAP-2-myr in the pTRA-IRES.GFP vector was introduced into tTA-BJAB cells. The infected GFP-positive cells were sorted and assayed for anti-IgM–induced CD69 expression (Fig. 5 A) and SLAP-2 protein expression (Fig. 5 C). A ∼5-fold upregulation of CD69 expression was observed in vector-infected cells after BCR cross-linking (data not shown). Expression of wild-type SLAP-2 suppressed anti-IgM–induced CD69 levels by ∼2-fold (Fig. 5 A). In contrast, anti-IgM–induced CD69 expression in cells infected with SLAP-2-myr was comparable to the vector control (Fig. 5 A). Although SLAP-2-myr was expressed at slightly lower levels than wild-type SLAP-2 (Fig. 5 C), the complete loss of activity when the putative myristoylation site was disrupted suggests that this motif may play an important role in SLAP-2 function in BJAB cells. Wild-type SLAP-2 and SLAP-2-myr were also expressed in tTA-Jurkat cells at approximately equal levels (Fig. 5 D) and their effects on TCR signaling were examined (Fig. 5 B). TCR cross-linking stimulated a ∼4.5-fold upregulation of CD69 expression in vector-infected cells (data not shown). Expression of wild-type SLAP-2 strongly blocked upregulation of CD69 in response to TCR cross-linking (Fig. 5 B). Once again, mutation of the myristoylation consensus sequence significantly compromised this activity, suggesting that this motif is also important for SLAP-2 function in T lymphocytes.


Functional cloning of Src-like adapter protein-2 (SLAP-2), a novel inhibitor of antigen receptor signaling.

Holland SJ, Liao XC, Mendenhall MK, Zhou X, Pardo J, Chu P, Spencer C, Fu A, Sheng N, Yu P, Pali E, Nagin A, Shen M, Yu S, Chan E, Wu X, Li C, Woisetschlager M, Aversa G, Kolbinger F, Bennett MK, Molineaux S, Luo Y, Payan DG, Mancebo HS, Wu J - J. Exp. Med. (2001)

The NH2-terminal myristoylation site and COOH-terminal unique regions of SLAP-2 are required for inhibition of antigen receptor signaling. Epitope-tagged wild-type SLAP-2, SLAP-2-myr, and SLAP-2-ΔC in the pTRA-IRES.GFP vector were stably introduced into tTA-BJAB or tTA-Jurkat cells, and GFP-positive cells were enriched by sorting. Induced surface CD69 expression was analyzed in vector-infected cells (dotted line) and cells infected with wild-type or mutant SLAP-2 (solid line) with analytical-gating on GFP expression. The geometric means of APC-CD69 fluorescence is shown. (A) tTA-BJAB cells stimulated with anti-IgM F(ab′)2.Wild-type (WT) SLAP-2 reduced anti-IgM F(ab′)2-induced CD69 expression by ∼2-fold, whereas SLAP-2-myr and SLAP-2-ΔC resembled the vector control. Percentage of GFP positive cells: vector 91%; wild-type SLAP-2 89%; SLAP-2-myr 80%; SLAP-2-ΔC 87%. (B) tTA-Jurkat cells stimulated with C305. Wild-type SLAP-2 reduced C305-induced CD69 expression by ∼3-fold, whereas inhibition was compromised in cells expressing SLAP-2-myr and SLAP-2-ΔC. Percentage of GFP-positive cells: vector 84%; wild-type SLAP-2 46%; SLAP-2-myr 44%; SLAP-2-ΔC 45%. (C) Equal aliquots of infected tTA-BJAB cells were lysed and analyzed by anti-FLAG Western blot for SLAP-2 protein expression. (D) Equal aliquots of infected tTA-Jurkat cells were lysed and analyzed by anti-FLAG Western blot for SLAP-2 protein expression. (E) Membrane pellet (P) and soluble fraction (S) of wild-type SLAP-2 and SLAP-2-myr–infected tTA-BJAB cells were immunoprecipitated (top panel) or loaded directly (center bottom panels) and immunoblotted with antibodies raised against the FLAG epitope (top panel), the integral membrane protein CD40 (center panel), and the cytoplasmic protein JNK (bottom panel). A fraction of wild-type SLAP-2 but not SLAP-2-myr was localized in the membrane fraction.
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Related In: Results  -  Collection

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fig5: The NH2-terminal myristoylation site and COOH-terminal unique regions of SLAP-2 are required for inhibition of antigen receptor signaling. Epitope-tagged wild-type SLAP-2, SLAP-2-myr, and SLAP-2-ΔC in the pTRA-IRES.GFP vector were stably introduced into tTA-BJAB or tTA-Jurkat cells, and GFP-positive cells were enriched by sorting. Induced surface CD69 expression was analyzed in vector-infected cells (dotted line) and cells infected with wild-type or mutant SLAP-2 (solid line) with analytical-gating on GFP expression. The geometric means of APC-CD69 fluorescence is shown. (A) tTA-BJAB cells stimulated with anti-IgM F(ab′)2.Wild-type (WT) SLAP-2 reduced anti-IgM F(ab′)2-induced CD69 expression by ∼2-fold, whereas SLAP-2-myr and SLAP-2-ΔC resembled the vector control. Percentage of GFP positive cells: vector 91%; wild-type SLAP-2 89%; SLAP-2-myr 80%; SLAP-2-ΔC 87%. (B) tTA-Jurkat cells stimulated with C305. Wild-type SLAP-2 reduced C305-induced CD69 expression by ∼3-fold, whereas inhibition was compromised in cells expressing SLAP-2-myr and SLAP-2-ΔC. Percentage of GFP-positive cells: vector 84%; wild-type SLAP-2 46%; SLAP-2-myr 44%; SLAP-2-ΔC 45%. (C) Equal aliquots of infected tTA-BJAB cells were lysed and analyzed by anti-FLAG Western blot for SLAP-2 protein expression. (D) Equal aliquots of infected tTA-Jurkat cells were lysed and analyzed by anti-FLAG Western blot for SLAP-2 protein expression. (E) Membrane pellet (P) and soluble fraction (S) of wild-type SLAP-2 and SLAP-2-myr–infected tTA-BJAB cells were immunoprecipitated (top panel) or loaded directly (center bottom panels) and immunoblotted with antibodies raised against the FLAG epitope (top panel), the integral membrane protein CD40 (center panel), and the cytoplasmic protein JNK (bottom panel). A fraction of wild-type SLAP-2 but not SLAP-2-myr was localized in the membrane fraction.
Mentions: Myristoylation has been shown to localize signaling proteins including Src family kinases to the membrane compartment and in many cases to be critical for their function (29). As SLAP-2 contains a putative NH2-terminal consensus motif (MGX1–4T/S) for addition of a myristoyl group, we generated an amino acid substitution in the myristoylation motif within a FLAG-tagged full length SLAP-2 (SLAP-2-myr) and examined its effect on antigen receptor–induced signaling. SLAP-2-myr in the pTRA-IRES.GFP vector was introduced into tTA-BJAB cells. The infected GFP-positive cells were sorted and assayed for anti-IgM–induced CD69 expression (Fig. 5 A) and SLAP-2 protein expression (Fig. 5 C). A ∼5-fold upregulation of CD69 expression was observed in vector-infected cells after BCR cross-linking (data not shown). Expression of wild-type SLAP-2 suppressed anti-IgM–induced CD69 levels by ∼2-fold (Fig. 5 A). In contrast, anti-IgM–induced CD69 expression in cells infected with SLAP-2-myr was comparable to the vector control (Fig. 5 A). Although SLAP-2-myr was expressed at slightly lower levels than wild-type SLAP-2 (Fig. 5 C), the complete loss of activity when the putative myristoylation site was disrupted suggests that this motif may play an important role in SLAP-2 function in BJAB cells. Wild-type SLAP-2 and SLAP-2-myr were also expressed in tTA-Jurkat cells at approximately equal levels (Fig. 5 D) and their effects on TCR signaling were examined (Fig. 5 B). TCR cross-linking stimulated a ∼4.5-fold upregulation of CD69 expression in vector-infected cells (data not shown). Expression of wild-type SLAP-2 strongly blocked upregulation of CD69 in response to TCR cross-linking (Fig. 5 B). Once again, mutation of the myristoylation consensus sequence significantly compromised this activity, suggesting that this motif is also important for SLAP-2 function in T lymphocytes.

Bottom Line: Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade.In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl.Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

View Article: PubMed Central - PubMed

Affiliation: Rigel, Incorporated, South San Francisco, CA 94080, USA. sholland@rigel.com

ABSTRACT
In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expressed in hematopoietic cells. Overexpression of SLAP-2 in B and T cell lines specifically impaired antigen receptor-mediated signaling events, including CD69 surface marker upregulation, nuclear factor of activated T cells (NFAT) promoter activation and calcium influx. Signaling induced by phorbol myristate acetate (PMA) and ionomycin was not significantly reduced, suggesting SLAP-2 functions proximally in the antigen receptor signaling cascade. The SLAP-2 protein contains an NH2-terminal myristoylation consensus sequence and SH3 and SH2 Src homology domains, but lacks a tyrosine kinase domain. In antigen receptor-stimulated cells, SLAP-2 associated with several tyrosine phosphorylated proteins, including the ubiquitin ligase Cbl. Deletion of the COOH terminus of SLAP-2 blocked function and abrogated its association with Cbl. Mutation of the putative myristoylation site of SLAP-2 compromised its inhibitory activity and impaired its localization to the membrane compartment. Our identification of the negative regulator SLAP-2 demonstrates that a retroviral-based screening strategy may be an efficient way to identify and characterize the function of key components of many signal transduction systems.

Show MeSH
Related in: MedlinePlus